Carr - CSF_Prion_TSQQuantiva_directMRM

Prion MRM assay development and clinical study

  • Organism: Homo sapiens, Mus musculus, Rattus norvegicus, Macaca cynomolgus
  • Instrument: TSQ Quantiva,Q Exactive
  • SpikeIn: Yes
  • Keywords: targeted mass spectrometry; multiple reaction monitoring mass spectrometry; prion; cerebrospinal fluid; biomarker
  • Lab head: Steve Carr
Abstract
Therapies currently in preclinical development for prion disease seek to lower prion protein (PrP) expression in the brain. Trials of such therapies are likely to rely on quantification of PrP in cerebrospinal fluid (CSF) as a pharmacodynamic biomarker and possibly as a trial endpoint. Studies using PrP ELISA kits have shown that CSF PrP is lowered in the symptomatic phase of disease, a potential confounder for reading out the effect of PrP-lowering drugs in symptomatic patients. Because misfolding or proteolytic cleavage could potentially render PrP undetectable by ELISA, we sought to establish an orthogonal method for CSF PrP quantification. We developed a multi-species targeted mass spectrometry method based on multiple reaction monitoring (MRM) of nine PrP tryptic peptides quantified by stable isotope dilution analysis. Analytical validation experiments showed intra-day and inter-day assay reproducibility coefficients of variation less than 15% (below the best practices threshold), 3 orders of dynamic linear range that encompass the entire expected range of PrP concentration in CSF, lower limits of detection near 10 ng/mL and a similar recovery response from both CSF and brain homogenate matrices. Critical for preclinical assay development, comparable assay performance was found for peptides unique to 4 species. In N=55 CSF samples from individuals referred to prion surveillance centers with rapidly progressive dementia, all six human PrP peptides, spanning the N- and C-terminal domains of PrP, were uniformly reduced in prion disease cases compared to individuals with non-prion diagnoses. Thus, lowered CSF PrP concentration in prion disease is a genuine result of the disease process and not merely an artifact of ELISA-based measurement. As a result, dose-finding studies for PrP lowering drugs may need to be conducted in pre-symptomatic at-risk individuals rather than in symptomatic patients. We provide a targeted mass spectrometry-based method suitable for preclinical and clinical quantification of CSF PrP as a tool for drug development.
Experiment Description
15N protein standard (32.5 fmol) was added to samples of CSF. Urea, TCEP and CHAPS were added to final concentration 6M, 20 mM and 0.03%, respectively. Samples were mixed at 800 RPM 37°C for 30 min. Samples were cooled to room temperature and incubated with 39 mM iodoacetamide 30 min in the dark. Samples were diluted to 900 mM Urea with 0.2 M Trizma pH 8.1 and trypsin (Promega V5113) was added to 1:50 (E:S) and incubated at 37°C overnight. Digestion was quenched with 5% formic acid. Digested samples were desalted using StageTips, 2 punches of Empore C18 held with an adaptor in a microcentrifuge tube and processed as follows: 50 uL 90% acetonitrile/0.1% TFA, 50 uL 0.1% TFA, addition of digestion in aliquots of 150 uL centrifuging after each load, wash twice with 50 uL 0.1% TFA, elute twice with 50 uL 40% acetonitrile/0.1% TFA and store frozen until MS analysis. Frozen samples were dried under vacuum centrifugation, resuspended in 12 µL 3% acetonitrile/5% acetic acid, vortexed for 5 min at RT, centrifuged 12,000 x g for 5 min and 10 µL were transferred into an HPLC vial (Waters 186000273). HPLC vials were centrifuged briefly (30 - 60s) at 1,200 x g to remove air bubbles and transferred into the nanoLC autosampler compartment set to 7°C. Samples were analyzed on a TSQ Quantiva triple quadrupole mass spectrometer installed with a Nanospray Flex source and Easy-nLC 1000 system (Thermo). Ion source was set to positive ion mode with capillary temperature of 300°C, spray voltage of 2,000 and sweep gas set to 0. The Easy-nLC 1000 system was primed with mobile phase A (3% acetonitrile / 0.1% formic acid), mobile phase B (90% acetonitrile / 0.1% formic acid). Samples were injected (2 µL, 20% of digested sample) onto a 0.075 mm ID PicoFrit (New Objective) column pulled to a 10 µm emitter and custom-packed to 20 cm with 1.9 µm 200Å C18-AQ Reprosil beads (Dr. Maisch). The LC gradient was 0% B to 30% B for 55 min, 30% B to 60% B in 5 min, 60% B to 90 % B in 1 min using a flow rate of 200 nL/min. Collision energies were optimized over 4 steps, 2.5 V per step in batches of less than 500 transitions per batch, 3 to 4 transitions were monitored per peptide in the final MRM method using a 1.5s cycle time.
Sample Description
Rat and cynomolgus monkey CSF were purchased from BioIVT. Human brain tissue was from a non-prion disease control individual provided by the National Prion Disease Pathology Surveillance Center (Cleveland, OH). Mouse brain tissue from Edinburgh PrP knockout mice backcrossed to a C57BL/10 background, and matching tissue from wild-type C57BL/10 mice were provided by Gregory J. Raymond (NIAID Rocky Mountain Labs, Hamilton, MT) Untagged recombinant HuPrP23-230 (MW=22,878) and MoPrP23-231 (MW=23,151), corresponding to full-length post-translationally modified human and mouse PrP without the signal peptide or GPI signal but retaining an N-terminal methionine, were purified by denaturation and Ni-NTA affinity from E. coli inclusion bodies as previously described (31, 32), using a vector generously provided by Byron Caughey (NIAID Rocky Mountain Labs, Hamilton, MT). 15N incorporation was achieved by growing the E. coli in 15N cell growth medium (Cambridge Isotope Laboratories CGM-1000-N) induced with 15N auto-induction medium (Millipore 71759-3).
Created on 7/29/19, 2:20 PM

­Domain-specific quantification of prion protein in cerebrospinal fluid by targeted mass spectrometry

Eric Vallabh Minikel, Eric Kuhn, Alexandra R Cocco, Sonia M Vallabh, Christina R Hartigan, Andrew G Reidenbach, Jiri G Safar, Gregory J Raymond, Michael D McCarthy, Rhonda O'Keefe, Franc Llorens, Inga Zerr, Sabina Capellari, Piero Parchi, Stuart L Schreiber, Steven A Carr

Abstract

Therapies currently in preclinical development for prion disease seek to lower prion protein (PrP) expression in the brain. Trials of such therapies are likely to rely on quantification of PrP in cerebrospinal fluid (CSF) as a pharmacodynamic biomarker and possibly as a trial endpoint. Studies using PrP ELISA kits have shown that CSF PrP is lowered in the symptomatic phase of disease, a potential confounder for reading out the effect of PrP-lowering drugs in symptomatic patients. Because misfolding or proteolytic cleavage could potentially render PrP undetectable by ELISA, we sought to establish an orthogonal method for CSF PrP quantification. We developed a multi-species targeted mass spectrometry method based on multiple reaction monitoring (MRM) of nine PrP tryptic peptides quantified by stable isotope dilution analysis. Analytical validation experiments showed intra-day and inter-day assay reproducibility coefficients of variation less than 15% (below the best practices threshold), 3 orders of dynamic linear range that encompass the entire expected range of PrP concentration in CSF, lower limits of detection near 10 ng/mL and a similar recovery response from  both CSF and brain homogenate matrices. Critical for preclinical assay development, comparable assay performance was found for peptides unique to 4 species. In N=55 CSF samples from individuals referred to prion surveillance centers with rapidly progressive dementia, all six human PrP peptides, spanning the N- and C-terminal domains of PrP, were uniformly reduced in prion disease cases compared to individuals with non-prion diagnoses. Thus, lowered CSF PrP concentration in prion disease is a genuine result of the disease process and not merely an artifact of ELISA-based measurement. As a result, dose-finding studies for PrP lowering drugs may need to be conducted in pre-symptomatic at-risk individuals rather than in symptomatic patients. We provide a targeted mass spectrometry-based method suitable for preclinical and clinical quantification of CSF PrP as a tool for drug development.

Samples

CSF samples for assay development were large volume normal pressure hydrocephalus samples provided by MIND Tissue Bank at Massachusetts General Hospital. Clinical CSF samples were premortem lumbar punctures from individuals referred to prion surveillance centers in Italy (Bologna) or Germany (Göttingen) with suspected prion disease and who were later either determined by autopsy or probable diagnostic criteria including real-time quaking-induced conversion as prion disease, or confirmed as non-prion cases on the basis of autopsy, patient recovery, or definitive other diagnostic test. Individuals with non-prion diagnoses (N=21) included autoimmune disease (N=8), non-prion neurodegenerative disease (N=6), psychiatric illness (N=3), stroke (N=1), brain cancer (N=1), and other (N=2). Sporadic prion disease cases (N=23) included probable cases (N=10) and autopsy-confirmed definite cases (N=13, of subtypes: 6 MM1, 3 VV2 and 4 other/unknown). Genetic prion disease cases (N=11) included D178N (N=2), E200K (N=7), and V210I (N=2). Samples were de-identified and broken into five batches (to be run on different days) randomly using an R script. Assay operators were blinded to diagnosis.

Rat and cynomolgus monkey CSF were purchased from BioIVT. Human brain tissue was from a non-prion disease control individual provided by the National Prion Disease Pathology Surveillance Center (Cleveland, OH). Mouse brain tissue from Edinburgh PrP knockout mice backcrossed to a C57BL/10 background, and matching tissue from wild-type C57BL/10 mice were provided by Gregory J. Raymond (NIAID Rocky Mountain Labs, Hamilton, MT)

Untagged recombinant HuPrP23-230 (MW=22,878) and MoPrP23-231 (MW=23,151), corresponding to full-length post-translationally modified human and mouse PrP without the signal peptide or GPI signal but retaining an N-terminal methionine, were purified by denaturation and Ni-NTA affinity from E. coli inclusion bodies as previously described (31, 32), using a vector generously provided by Byron Caughey (NIAID Rocky Mountain Labs, Hamilton, MT). 15N incorporation was achieved by growing the E. coli in 15N cell growth medium (Cambridge Isotope Laboratories CGM-1000-N) induced with 15N auto-induction medium (Millipore 71759-3).

Sample Preparation

15N protein standard (32.5 fmol) was added to samples of CSF. Urea, TCEP and CHAPS were added to final concentration 6M, 20 mM and 0.03%, respectively. Samples were mixed at 800 RPM 37°C for 30 min. Samples were cooled to room temperature and incubated with 39 mM iodoacetamide 30 min in the dark. Samples were diluted to 900 mM Urea with 0.2 M Trizma pH 8.1 and trypsin (Promega V5113) was added to 1:50 (E:S) and incubated at 37°C overnight. Digestion was quenched with 5% formic acid. Digested samples were desalted using StageTips, 2 punches of Empore C18 held with an adaptor in a microcentrifuge tube and processed as follows: 50 uL 90% acetonitrile/0.1% TFA, 50 uL 0.1% TFA, addition of digestion in aliquots of 150 uL centrifuging after each load, wash twice with 50 uL 0.1% TFA, elute twice with 50 uL 40% acetonitrile/0.1% TFA and store frozen until MS analysis.

LC-MS/MS Analysis

Samples of dried digested recombinant proteins or human cerebrospinal fluid were reconstituted in 3% acetonitrile/5% acetic acid to a final concentration of approximately 1 µg total protein per 1 µL and analyzed in a single injection using a standard 2h reversed-phase gradient. LC-MS/MS was performed using a QExactive Plus mass spectrometer (Thermo) equipped with a Proxeon Easy-nLC 1200 and a custom built nanospray source (James A. Hill Instrument Services). Samples were injected (1 to 2 µg) onto a 75 um ID PicoFrit column (New Objective) packed to 20 cm with Reprosil-Pur C18 AQ 1.9 um media (Dr. Maisch) and heated to 50°C. MS source conditions were set as follows: spray voltage 2000, capillary temperature 250°C, S-lens RF level 50. A single Orbitrap MS scan from 300 to 1800 m/z at a resolution of 70,000 with AGC set at 3e6 was followed by up to 12 MS/MS scans at a resolution of 17,500 with AGC set at 5e4. MS/MS spectra were collected with normalized collision energy of 25 and isolation width of 2.5 amu. Dynamic exclusion was set to 20 s and peptide match was set to preferred. Mobile phases consisted of 3% acetonitrile/0.1% formic acid as solvent A, 90% acetonitrile/0.1% formic acid as solvent B. Flow rate was set to 200 nL/min throughout the gradient, 2% - 6% B in 1 min, 6% - 30% B in 84 min, 30% - 60% B in 9 min, 60% - 90% B in 1 min with a hold at 90% B for 5 min.

MS: QExactive Plus (Thermo)

LC: Proxeon Easy-nLC 1200 (Thermo)

Source: Custom Jaime Hill Source

Column: PicoFrit (New Objective) 0.075 x 200 mm

Media: Reprosil-Pur C18 AQ 1.9 um (Dr. Maisch)

Column temperature:  50°C (Phoenix S&T)

MS source conditions: spray voltage 2000, ion transfer tube temperature 250°C, S-lens RF level 50

MS scans: Orbitrap MS scan from 300 to 1800 m/z at a resolution of 70,000 with AGC set at 3e6

MS/MS scans: top 12 MS/MS, Resolution 17,500, AGC 5e4

NCE: 25

Isolation width: 2.5 amu

Dynamic exclusion 20 s

Database: UniProt.human.20141017.RNFISnr_allspeciesprion

Mobile phase A: 3% acetonitrile/0.1% formic acid

Mobile phase B: 90% acetonitrile/0.1% formic acid

Flow rate: 200 nL/min

Gradient: 2% - 6% B in 1 min, 6% - 30% B in 84 min, 30% - 60% B in 9 min, 60% - 90% B in 1 min with a hold at 90% B for 5 min

 

MS Database Search

MS data were analyzed using Spectrum Mill MS Proteomics Workbench software Rev B.06.01.202 (Agilent Technologies). Similar MS/MS spectra acquired on the same precursor m/z within +/- 60 sec were merged. MS/MS spectra were excluded from searching if they failed the quality filter by not having a sequence tag length > 0 (i.e., minimum of two masses separated by the in-chain mass of an amino acid) or did not have a precursor MH+ in the range of 600-6000. All extracted spectra were searched against a UniProt database containing human and mouse reference proteome sequences downloaded from the UniProt web site on October 17, 2014 with redundant sequences removed. A set of common laboratory contaminant proteins (150 sequences) were appended to this database and verified to contain the sequences for human and mouse major prion protein. Search parameters included: ESI-QEXACTIVE-HCD-v2 scoring, parent and fragment mass tolerance of 20 ppm, 40% minimum matched peak intensity and ‘trypsin’ enzyme specificity up to 2 missed cleavages. Fixed modification was carbamidomethylation at cysteine and variable modifications were oxidized methionine, deamidation of asparagine and pyro-glutamic acid. Database matches were autovalidated at the peptide and protein level in a two-step process with identification FDR estimated by target-decoy-based searches using reversed sequences. The list of identified proteins was further filtered to contain proteins and protein isoforms with at least 2 unique peptides and an aggregate protein score greater than 20. Protein-peptide comparison report comprised of all validated peptides was exported which included a ranked summary by intensity of all peptides unique to prion protein.

 

LC-MRM-MS Analysis

Frozen samples were dried under vacuum centrifugation, resuspended in 12 µL 3% acetonitrile/5% acetic acid, vortexed for 5 min at RT, centrifuged 12,000 x g for 5 min and 10 µL were transferred into an HPLC vial (Waters 186000273). HPLC vials were centrifuged briefly (30 - 60s) at 1,200 x g to remove air bubbles and transferred into the nanoLC autosampler compartment set to 7°C. Samples were analyzed on a TSQ Quantiva triple quadrupole mass spectrometer installed with a Nanospray Flex source and Easy-nLC 1000 system (Thermo). Ion source was set to positive ion mode with capillary temperature of 300°C, spray voltage of 2,000 and sweep gas set to 0. The Easy-nLC 1000 system was primed with mobile phase A (3% acetonitrile / 0.1% formic acid), mobile phase B (90% acetonitrile / 0.1% formic acid). Samples were injected (2 µL, 20% of digested sample) onto a 0.075 mm ID PicoFrit (New Objective) column pulled to a 10 µm emitter and custom-packed to 20 cm with 1.9 µm 200Å C18-AQ Reprosil beads (Dr. Maisch). The LC gradient was 0% B to 30% B for 55 min, 30% B to 60% B in 5 min, 60% B to 90 % B in 1 min using a flow rate of 200 nL/min.  Collision energies were optimized over 4 steps, 2.5 V per step in batches of less than 500 transitions per batch, 3 to 4 transitions were monitored per peptide in the final MRM method (see transitions listed below) using a 1.5s cycle time, including transitions that correspond to the oxidized methionine version of peptide VVEQMCITQYER.

MS: Quantiva (Thermo)

LC: Proxeon Easy-nLC 1000 (Thermo)

Source: Nanospray Flex (Thermo)

Column: PicoFrit (New Objective) 0.075 x 200 mm

Media: Reprosil-Pur C18 AQ 1.9 um (Dr. Maisch)

Column temperature:  50°C (Phoenix S&T)

MS source conditions: spray voltage 2000, ion transfer tube temperature 300°C, sweep gas 0

MRM: See transition table

Background proteome: human.protdb, Macaca_fascicularis.protdb,

Spectral library: PrP_mouhum.blib

Collision Energy library: PrP.optdb

Mobile phase A: 3% acetonitrile/0.1% formic acid

Mobile phase B: 90% acetonitrile/0.1% formic acid

Flow rate: 200 nL/min

Gradient: 2% - 6% B in 1 min, 6% - 30% B in 84 min, 30% - 60% B in 9 min, 60% - 90% B in 1 min with a hold at 90% B for 5 min

MRM Data Analysis

Extracted Ion chromatograms (XIC) of all transition ions were verified and integrated using a Skyline document as described (39) (Skyline version 4.1.0.11796, https://brendanx-uw1.gs.washington.edu/labkey/project/home/software/Skyline/begin.view) that contained the sequences, spectral libraries derived from LC-MS/MS of the 15N/13C-labeled synthetic heavy peptide and background proteome of human, mouse and macaca fascicularis. After peak integration, the Skyline report file was exported as a text delimited file with peak area columns labeled as “Light”, “Heavy” or “15N”. The single most intense, interference-free, reproducibly measured transition (see Table) were used for quantification and statistical analysis. Report included columns: Protein Name, Protein Gene, Protein Species, Peptide Sequence, Peptide Modified Sequence, File Name, Acquired Time, Replicate Name, SampleGroup, Peptide Retention Time, Precursor Mz, Fragment Ion, Area, Area Ratio, Total Area, Total Area Ratio.

Skyline Report: Peptide Totals PrionProject.skyr

 

Metadata

Samples: Recombinant protein

  1. Human PrP
  2. Mouse PrP
  3. 15N(U) human PrP

Samples: Brain Homogenates

  1. Mouse
  2. Human

Samples: CSF

  1. Human control
  2. Cynomolgus monkey
  3. Rat
  4. Clinical patients (N=55) incl. one interplate control (IPC)

 

Supplementary Table 1. Precursor and product ions monitored by LC-MRM-MS for prion peptides.

 

peptide modified sequence

precursor charge

fragment ion

product charge

precursor mz          [light]

precursor mz       [heavy]

precursor mz

[15N]

product mz

[light]

product mz [heavy]

product mz

[15n]

best fragment ion

PIIHFGSDYEDR

3

b4

1

483.57

486.90

489.21

461.29

461.29

467.27

 

3

y9

2

483.57

486.90

489.21

563.23

568.24

570.21

 

3

y4

1

483.57

486.90

489.21

582.25

592.26

589.23

y4

3

y7

1

483.57

486.90

489.21

841.33

851.34

851.30

 

RPKPGGWNTGGSR

3

y4

1

457.24

460.58

464.55

376.19

386.20

383.17

y4

3

y5

1

457.24

460.58

464.55

477.24

487.25

485.22

 

3

y6

1

457.24

460.58

464.55

591.28

601.29

601.25

 

YPGQGSPGGNR

2

y5

1

545.26

550.26

553.23

500.26

510.27

509.23

y5

2

y7

1

545.26

550.26

553.23

644.31

654.32

655.28

 

2

y9

1

545.26

550.26

553.23

829.39

839.40

843.35

 

GENFTETDVK

2

y4

1

570.26

574.27

576.25

462.26

470.27

467.24

 

2

y5

1

570.26

574.27

576.25

591.30

599.31

597.28

 

2

y6

1

570.26

574.27

576.25

692.35

700.36

699.33

 

2

y7

1

570.26

574.27

576.25

839.41

847.43

847.39

 

VVEQMC[+57]ITQYER

2

y5

1

778.37

783.37

786.84

696.33

706.34

705.30

y5

2

y7

1

778.37

783.37

786.84

969.45

979.45

980.41

 

2

y8

1

778.37

783.37

786.84

1100.49

1110.49

1112.45

 

VVEQM[+16]C[+57]ITQYER

2

y5

1

786.36

791.37

794.84

696.33

706.34

705.30

y5

2

y7

1

786.36

791.37

794.84

969.45

979.45

980.41

 

2

y8

1

786.36

791.37

794.84

1116.48

1126.49

1128.45

 

ESQAYYQR

2

y3

1

522.74

527.75

529.22

466.24

476.25

473.22

y3

2

y4

1

522.74

527.75

529.22

629.30

639.31

637.28

 

2

y5

1

522.74

527.75

529.22

700.34

710.35

709.31

 

VVEQMC[+57]VTQYQK

2

y5

1

756.86

760.87

n/a

667.34

675.36

n/a

y5

2

y7

1

756.86

760.87

n/a

926.44

934.45

n/a

 

2

y8

1

756.86

760.87

n/a

1057.48

1065.49

n/a

 

VVEQM[+16]C[+57]VTQYQK

2

y5

1

764.86

768.87

n/a

667.34

675.36

n/a

y5

2

y7

1

764.86

768.87

n/a

926.44

934.45

n/a

 

2

y8

1

764.86

768.87

n/a

1073.48

1081.49

n/a

 

ESQAYYDGR

2

y4

1

544.74

549.74

n/a

510.23

520.24

n/a

y4

2

y5

1

544.74

549.74

n/a

673.29

683.30

n/a

 

2

y6

1

544.74

549.74

n/a

744.33

754.34

n/a

 

VVEQMC[+57]ITQYEK

2

y5

1

764.36

768.37

n/a

668.32

676.34

n/a

y5

2

y7

1

764.36

768.37

n/a

941.44

949.45

n/a

 

2

y8

1

764.36

768.37

n/a

1072.48

1080.49

n/a

 

VVEQM[+16]C[+57]ITQYEK

2

y5

1

772.36

776.37

n/a

668.32

676.34

n/a

y5

2

y7

1

772.36

776.37

n/a

941.44

949.45

n/a

 

2

y8

1

772.36

776.37

n/a

1088.48

1096.49

n/a

 

 

Datafiles:

 

Experiment

Skyline file

Datafiles

PrP_MSMS_15N Isotopic Purity

n.a.

FA20160907_EK_v1187_NPH_CSF_30ul_01

FA20160907_EK_v1187_NPH_CSF_30ul_02

FR20160913_EK_humanPrP_01

FR20160913_EK_mousePrP_01

HZ20180806_ARC_15NPrP_1ug_02_01

HZ20180806_ARC_15NPrP_1ug_02_02

PrP_MRM_Exp6_Species Specificity

PrP_MRM_Exp6_final_171030

QQ171027_CSF_Hh_02

QQ171027_CSF_Jj_4_01

QQ171027_CSF_K_01

QQ171027_CSF_L_01

QQ171027_CSF_M_01

QQ171027_CSF_N_01

QQ171027_CSF_Oo_02

PrP_MRM_Exp7_Matrix Detectability

PrP_MRM_Exp7_final_171109

QQ171103_CSF_1_02

QQ171103_CSF_2_02

QQ171103_CSF_3_02

QQ171103_CSF_4_02

PrP_MRM_Exp8_15N Response Curves

 

PrP_MRM_Exp8_final_180103

QQ171228_PrP_curve1_0ngmL_01

QQ171228_PrP_curve1_25ngmL_01

QQ171228_PrP_curve1_50ngmL_01

QQ171228_PrP_curve1_100ngmL_01

QQ171228_PrP_curve1_106ngmL_01

QQ171228_PrP_curve1_200ngmL_01

QQ171228_PrP_curve1_301ngmL_01

QQ171228_PrP_curve1_400ngmL_01

QQ171228_PrP_curve1_603ngmL_01

QQ171228_PrP_curve1_800ngmL_01

QQ171228_PrP_curve1_1205ngmL_01

QQ171228_PrP_curve1_blank_01

QQ171228_PrP_curve1_blank_02

QQ171228_PrP_curve1_carryover_01

QQ171228_PrP_curve1_carryover_02

QQ171228_PrP_curve2_0ngmL_01

QQ171228_PrP_curve2_25ngmL_01

QQ171228_PrP_curve2_50ngmL_01

QQ171228_PrP_curve2_100ngmL_01

QQ171228_PrP_curve2_106ngmL_01

QQ171228_PrP_curve2_200ngmL_01

QQ171228_PrP_curve2_301ngmL_01

QQ171228_PrP_curve2_400ngmL_01

QQ171228_PrP_curve2_603ngmL_01

QQ171228_PrP_curve2_800ngmL_01

QQ171228_PrP_curve2_1205ngmL_01

QQ171228_PrP_curve2_blank_01

QQ171228_PrP_curve2_blank_02

QQ171228_PrP_curve2_carryover_01

QQ171228_PrP_curve2_carryover_02

PrP_MRM_Exp9_Accuracy_MouseBrain

 

PrP_MRM_Exp9_final_180116

QQ171228_PrP_Exp9_A1_01

QQ171228_PrP_Exp9_A2_01

QQ171228_PrP_Exp9_B1_01

QQ171228_PrP_Exp9_B2_01

QQ171228_PrP_Exp9_C1_01

QQ171228_PrP_Exp9_C2_01

QQ171228_PrP_Exp9_D1_01

QQ171228_PrP_Exp9_D2_01

QQ171228_PrP_Exp9_E1_01

QQ171228_PrP_Exp9_E2_01

QQ171228_PrP_Exp9_F1_01

QQ171228_PrP_Exp9_F2_01

QQ171228_PrP_Exp9_G1_01

QQ171228_PrP_Exp9_G2_01

QQ171228_PrP_Exp9_IPC_01_01

QQ171228_PrP_Exp9_IPC_02_01

PrP_MRM_Exp10_Dynamic Range

 

PrP_MRM_Exp10_final_180430

QQ180404_PrP_Exp10_A1_01

QQ180404_PrP_Exp10_A2_01

QQ180404_PrP_Exp10_A3_01

QQ180404_PrP_Exp10_A4_01

QQ180404_PrP_Exp10_A5_01

QQ180404_PrP_Exp10_B1_01

QQ180404_PrP_Exp10_B2_01

QQ180404_PrP_Exp10_B3_01

QQ180404_PrP_Exp10_B4_01

QQ180404_PrP_Exp10_B5_01

QQ180404_PrP_Exp10_BlankCSF_01

QQ180404_PrP_Exp10_C1_01

QQ180404_PrP_Exp10_C2_01

QQ180404_PrP_Exp10_C3_01

QQ180404_PrP_Exp10_C4_01

QQ180404_PrP_Exp10_C5_01

QQ180404_PrP_Exp10_D1_01

QQ180404_PrP_Exp10_D2_01

QQ180404_PrP_Exp10_D3_01

QQ180404_PrP_Exp10_D4_01

QQ180404_PrP_Exp10_D5_01

QQ180404_PrP_Exp10_IPC1_01 QQ180404_PrP_Exp10_IPC2_01

QQ180404_PrP_Exp10_Zero_01

PrP_MRM_Exp11_Spike Recovery

PrP_MRM_Exp11_final_180601

QQ180508_PrP_Exp11_39_1_02

QQ180508_PrP_Exp11_39_2_02

QQ180508_PrP_Exp11_39_3_02

QQ180508_PrP_Exp11_47_1_02

QQ180508_PrP_Exp11_47_2_02

QQ180508_PrP_Exp11_47_3_02

QQ180508_PrP_Exp11_A000_01_02

QQ180508_PrP_Exp11_A000_02_02

QQ180508_PrP_Exp11_A025_01_02

QQ180508_PrP_Exp11_A025_02_02

QQ180508_PrP_Exp11_A050_01_02

QQ180508_PrP_Exp11_A050_02_02

QQ180508_PrP_Exp11_A075_01_02

QQ180508_PrP_Exp11_A075_02_02

QQ180508_PrP_Exp11_A100_01_02

QQ180508_PrP_Exp11_A100_02_02

QQ180508_PrP_Exp11_IPC_01_02

QQ180508_PrP_Exp11_IPC_02_02

PrP_MRM_Exp12_Precision

PrP_MRM_Exp12_final_180406

QQ180327_PrP_Exp12_H1_01

QQ180327_PrP_Exp12_H2_01

QQ180327_PrP_Exp12_H3_01

QQ180327_PrP_Exp12_H4_01

QQ180327_PrP_Exp12_H5_01

QQ180327_PrP_Exp12_L1_01

QQ180327_PrP_Exp12_L2_01

QQ180327_PrP_Exp12_L3_01

QQ180327_PrP_Exp12_L4_01

QQ180327_PrP_Exp12_L5_01

QQ180327_PrP_Exp12_M1_01

QQ180327_PrP_Exp12_M2_01

QQ180327_PrP_Exp12_M3_01

QQ180327_PrP_Exp12_M4_01

QQ180327_PrP_Exp12_M5_01

QQ180404_PrP_Exp12_IPC_01

QQ180404_PrP_Exp12_Rat9_01

PrP_MRM_Exp13_Plastic&Formulation

PrP_MRM_Exp13_final_180618

QQ180510_PrP_Exp13_C0A_01

QQ180510_PrP_Exp13_C0B_02

QQ180510_PrP_Exp13_C1A_01

QQ180510_PrP_Exp13_C1B_02

QQ180510_PrP_Exp13_C2A_01

QQ180510_PrP_Exp13_C2B_02

QQ180510_PrP_Exp13_C3A_01

QQ180510_PrP_Exp13_C3B_02

QQ180510_PrP_Exp13_N0A_01

QQ180510_PrP_Exp13_N0B_02

QQ180510_PrP_Exp13_N1A_01

QQ180510_PrP_Exp13_N1B_02

QQ180510_PrP_Exp13_N2A_01

QQ180510_PrP_Exp13_N2B_02

QQ180510_PrP_Exp13_N3A_01

QQ180510_PrP_Exp13_N3B_02

PrP_MRM_Exp14_15N ProteinStd Pilot

PrP_MRM_Exp14_final_180724

QC180724_PrP_Exp14_A_02

QC180724_PrP_Exp14_B_02

QC180724_PrP_Exp14_C_02

QC180724_PrP_Exp14_D_01

QC180724_PrP_Exp14_E_01

QC180724_PrP_Exp14_F_01

PrP_MRM_Exp15_Dilution Linearity

PrP_MRM_Exp15_final_180802

QC180731_PrP_Exp15_A_01

QC180731_PrP_Exp15_B_01

QC180731_PrP_Exp15_C_01

QC180731_PrP_Exp15_D_01

QC180731_PrP_Exp15_E_01

QC180731_PrP_Exp15_F_01

QC180731_PrP_Exp15_G_01

QC180731_PrP_Exp15_H_01

QC180731_PrP_Exp15_I_01

QC180731_PrP_Exp15_J_01

QC180731_PrP_Exp15_K_01

QC180731_PrP_Exp15_L_01

PrP_MRM_Exp16_Clinical Samples Set 1

PrP_MRM_Exp16_final_190115

QZ180906_PrP_Exp16_Set1_M1_01

QZ180906_PrP_Exp16_Set1_N1_01

QZ180906_PrP_Exp16_Set1_O1_01

QZ180910_PrP_Exp16_Set1_C1_03

QZ180910_PrP_Exp16_Set1_D1_02

QZ180910_PrP_Exp16_Set1_E1_02

QZ180910_PrP_Exp16_Set1_F1_02

QZ180910_PrP_Exp16_Set1_G1_02

QZ180910_PrP_Exp16_Set1_H1_02

QZ180910_PrP_Exp16_Set1_I1_02

QZ180910_PrP_Exp16_Set1_J1_02

QZ180910_PrP_Exp16_Set1_K1_02

QZ180910_PrP_Exp16_Set1_L1_02

QZ180911_PrP_Exp16_Set1_P1_02

QZ180911_PrP_Exp16_Set1_Q1_02

QZ180911_PrP_Exp16_Set1_R1_02

QZ180911_PrP_Exp16_Set1_S1_02

QZ180911_PrP_Exp16_Set1_T1_02

QZ180911_PrP_Exp16_Set1_U1_02

QZ180911_PrP_Exp16_Set1_V1_02

QZ180911_PrP_Exp16_Set1_X1_02

QZ180912_PrP_Exp16_Set1_W1_03

QZ180913_PrP_Exp16_Set1_A1_03

QZ180913_PrP_Exp16_Set1_B1_03

PrP_MRM_Exp17_Clinical Samples Set 2

PrP_MRM_Exp17_final_190115

QQ180928_PrP_Exp17_A2_01

QQ180928_PrP_Exp17_B2_01

QQ180928_PrP_Exp17_C2_01

QQ180928_PrP_Exp17_D2_01

QQ180928_PrP_Exp17_E2_01

QQ180928_PrP_Exp17_F2_01

QQ180928_PrP_Exp17_G2_01

QQ180928_PrP_Exp17_H2_01

QQ180928_PrP_Exp17_I2_01

QQ180928_PrP_Exp17_J2_01

QQ180928_PrP_Exp17_K2_01

QQ180928_PrP_Exp17_L2_01

QQ180928_PrP_Exp17_M2_01

QQ180928_PrP_Exp17_N2_01

QQ180928_PrP_Exp17_O2_01

QQ180928_PrP_Exp17_P2_01

QQ180930_PrP_Exp17_Q2_01

QQ180930_PrP_Exp17_R2_01

QQ180930_PrP_Exp17_S2_01

QQ180930_PrP_Exp17_T2_01

QQ180930_PrP_Exp17_U2_01

QQ180930_PrP_Exp17_V2_01

QQ180930_PrP_Exp17_W2_01

QQ180930_PrP_Exp17_X2_01

PrP_MRM_Exp18_Clinical Samples Set 3

PrP_MRM_Exp18_final_190115

QQ181107_PrP_Exp18_A3_03

QQ181107_PrP_Exp18_B3_03

QQ181116_PrP_Exp18_C3_03

QQ181116_PrP_Exp18_D3_03

QQ181116_PrP_Exp18_E3_03

QQ181116_PrP_Exp18_F3_03

QQ181116_PrP_Exp18_G3_04

QQ181116_PrP_Exp18_H3_03

QQ181116_PrP_Exp18_I3_03

QQ181116_PrP_Exp18_J3_03

QQ181116_PrP_Exp18_K3_03

QQ181116_PrP_Exp18_L3_03

QQ181116_PrP_Exp18_M3_03

QQ181116_PrP_Exp18_N3_03

QQ181116_PrP_Exp18_O3_03

QQ181116_PrP_Exp18_P3_03

QQ181116_PrP_Exp18_Q3_03

QQ181116_PrP_Exp18_R3_03

QQ181116_PrP_Exp18_S3_03

QQ181116_PrP_Exp18_T3_03

QQ181116_PrP_Exp18_U3_03

QQ181116_PrP_Exp18_V3_03

QQ181116_PrP_Exp18_W3_03

QQ181116_PrP_Exp18_X3_03

PrP_MRM_Exp19_Clinical Samples Set 4

PrP_MRM_Exp19_final_190115

QQ181103_PrP_Exp19_A4_02

QQ181103_PrP_Exp19_B4_02

QQ181103_PrP_Exp19_C4_02

QQ181103_PrP_Exp19_D4_02

QQ181103_PrP_Exp19_E4_02

QQ181103_PrP_Exp19_F4_02

QQ181103_PrP_Exp19_G4_02

QQ181103_PrP_Exp19_H4_02

QQ181103_PrP_Exp19_I4_02

QQ181103_PrP_Exp19_J4_02

QQ181103_PrP_Exp19_K4_02

QQ181103_PrP_Exp19_L4_02

QQ181105_PrP_Exp19_M4_02

QQ181105_PrP_Exp19_N4_02

QQ181105_PrP_Exp19_O4_02

QQ181105_PrP_Exp19_P4_02

QQ181105_PrP_Exp19_Q4_02

QQ181105_PrP_Exp19_R4_02

QQ181105_PrP_Exp19_S4_02

QQ181105_PrP_Exp19_T4_02

QQ181105_PrP_Exp19_U4_02

QQ181105_PrP_Exp19_V4_02

QQ181105_PrP_Exp19_W4_02

QQ181105_PrP_Exp19_X4_02

PrP_MRM_Exp20_Clinical Samples Set 5

PrP_MRM_Exp20_final_190115

QQ181101_PrP_Exp20_A5_01

QQ181101_PrP_Exp20_B5_01

QQ181101_PrP_Exp20_C5_01

QQ181101_PrP_Exp20_D5_01

QQ181101_PrP_Exp20_E5_01

QQ181101_PrP_Exp20_F5_01

QQ181101_PrP_Exp20_G5_01

QQ181101_PrP_Exp20_H5_01

QQ181101_PrP_Exp20_I5_01

QQ181101_PrP_Exp20_J5_01

QQ181101_PrP_Exp20_K5_01

QQ181101_PrP_Exp20_L5_01

QQ181102_PrP_Exp20_M5_01

QQ181102_PrP_Exp20_N5_01

QQ181102_PrP_Exp20_O5_01

QQ181102_PrP_Exp20_P5_01

QQ181102_PrP_Exp20_Q5_01

QQ181102_PrP_Exp20_R5_01

QQ181102_PrP_Exp20_S5_01

QQ181102_PrP_Exp20_T5_01

QQ181102_PrP_Exp20_U5_01

QQ181102_PrP_Exp20_V5_01

QQ181102_PrP_Exp20_W5_01

QQ181102_PrP_Exp20_X5_01

 

 

This data is available under the CC BY 4.0 license.